Project Summary and Relevance Application of genome editing based therapy requires efficient delivery of editing agents into disease-relevant tissues and cells. Identification of novel delivery materials targeting somatic cells will greatly facilitate the advance of editing based therapy strategies to clinic. We propose to screen a large library of novel lipid nanoparticles for RNP (ribonucleoprotein) delivery of editing agents into the mammalian sensory organ inner ear. Inner ear is an ideal sensory organ to develop new delivery strategies. It consists of multiple differentiated somatic cell types without effective delivery options. Gene mutations in the major inner ear cell types have been associated with genetic hearing loss, which affects one in 500 newborns and currently has no effective therapies. Lipid-based nanoparticle carriers have emerged as one of the most promising materials for delivery and have been successfully used in clinical applications. We have developed a combinatorial library approach to synthesize degradable lipid-like nanoparticles under reductive intracellular environments, and capable of delivering biomolecules with high efficiency and low toxicity. The new bioreducible lipid nanoparticles (bLNPs) have been used to deliver genome editing agents with high efficiency and low toxicity in vivo. We have delivered genome editing RNP by cationic liposomes into mammalian inner ear in vivo, and rescued hearing in mouse models of human genetic hearing loss. To develop editing based therapies to treat diverse forms of genetic hearing loss, it is essential to develop a delivery strategy to target multiple inner ear cell types simultaneously. The mammalian inner has a complicated structure with multiple cell types in small numbers, making it particularly challenging to screen a delivery technology by conventional high-throughput strategies. The lack of a method to detect editing at the level of the individual cell type further hinders our ability to apply this technology in wildtype large animal models that are essential for development of this therapy for clinical application. By combining our strategies to screen nanoparticles for delivery of editing materials to X-linked genes in the male mouse inner ears in vivo, we will overcome these hurdles for effective delivery and editing in diverse inner ear cell types. The study of the human inner ear tissues ex vivo will provide evidence of the relevance of nanoparticle delivery in human disease-relevant tissues. Expansion of our work to large animal models will be a major step towards clinical application of this technology. Our approach with nanoparticles can be applied to the study in other organs requiring somatic cell type editing and in wildtype large animals.